Dengue virus (DENV), including the four serotypes, DENV-1, -2, -3, and -4, is a member in the family of Flaviviridae and listed as one of the NIAID Category A priority pathogens. Due to the lack of vaccine and antiviral drugs, dengue disease is now a major public health burden around the world. Up-to-date, there was no high throughput screening (HTS) amenable, robust and reliable assay(s) developed to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR) against DENV, which hindered the discovery and development of antiviral drugs. Here, we showed the development, optimization and validation of a HTS amenable, CPE-based assay against DENV-2, with Z'-value of 0.71, coefficient of variation (CV) of 6.3% and signal-to-background (S/B) ratio of 6.88 in 96-well plate. This assay was also miniaturized into 384- well plate format with comparable assay robustness. We have also validated the assay using the Library of Pharmacologically Active Compounds (LOPAC1280, Sigma-Aldrich, St. Louis, MO). This assay is now ready to be transferred to the designated Molecular Libraries Probe Production Centers Network (MLPCN) to screen the NIH MLSMR. All "hits" from the primary HTS will be further confirmed, validated and prioritized using assays available including the dose-response confirmatory assay, replicon-based secondary assay, and time-of-addition assay. Our long term goal is to develop novel antivirals against dengue disease in humans. The objectives of this application will be achieved by carrying out the following two Specific Aims: Specific Aim 1: To transfer the HTS amenable, CPE-based assay against DENV-2 to the designated HTS center to screen the NIH MLSMR. Specific Aim 2: To confirm, validate and prioritized hits from the primary HTS using assays including the dose-response assay, the replicon-based assay and time-of-addition assay. PUBLIC HEALTH RELEVANCE: Dengue virus (DENV) is a member in the family of Flaviviridae and one of the NIAID category A priority pathogens. With more than one-third of the world's population living in areas at risk for transmission, dengue infection is a leading cause of illness and death in the tropics and subtropics. As many as 100 million people are infected yearly, with an estimated 500,000 cases of severe dengue requiring hospitalization, of which a very large proportion is in children, with case fatality up to 5%. In the Americas as of December 14, 2009, there were 893,453 reported cases, including 23,980 Dengue hemorrhagic fever (DHF) cases and 370 deaths. Currently, there is no efficient vaccine and effective anti-viral drugs against Dengue diseases, which makes Dengue disease a huge public health burden.